1. Field of the Invention
The present invention relates generally to the field of the purification of nucleic acids. In particular, it relates to the purification of nucleic acids, for example, RNA, DNA, and/or PNA using polymer-modified surfaces and/or resin-based surfaces.
2. Description of Related Art
Many molecular biological techniques such as reverse transcription, cloning, restriction analysis, and sequencing involve the processing or analysis of biological materials. The materials most commonly analyzed in this regard are polynucleotides such as RNA and DNA. These techniques generally require that such materials be substantially free of contaminants capable of interfering with such processing or analysis procedures. Such contaminants generally include substances that block or inhibit chemical reactions, (e.g., nucleic acid or protein hybridizations, enzymatically catalyzed reactions, and other types of reactions, used in molecular biological techniques), substances that catalyze the degradation or depolymerization of a nucleic acid or other biological material of interest, or substances that provide “background” indicative of the presence in a sample of a quantity of a biological target material of interest when the nucleic acid is not, in fact present in the sample. Contaminants also include macromolecular substances from the in vivo or in vitro medium from which a nucleic acid material of interest is isolated, macromolecular substances such as enzymes, other types of proteins, polysaccharides, or polynucleotides, as well as lower molecular weight substances, such as lipids, low molecular weight enzyme inhibitors or nucleotides or oligonucleotides. Contaminants can also be introduced into a target biological material from chemicals or other materials used to isolate the material from other substances. Common contaminants of this last type include trace metals, dyes, and organic solvents.
Obtaining polynucleotides sufficiently free of contaminants for molecular biological applications is complicated by the complex systems in which such polynucleotides are typically found. These systems, e.g., cells from tissues, cells from body fluids such as blood, lymph, milk, saliva, mucus, urine, feces, semen, or the like, cells in culture, agarose or polyacrylamide gels, or solutions in which target nucleic acid amplification has been carried out, typically include significant quantities of contaminants from which the polynucleotide(s) of interest must be isolated before being used in a molecular biological procedure.
A variety of techniques have been developed to allow for the purification of polynucleotides from contaminants. Some of these techniques involve adsorbing nucleic acids on glass or silica-gel particles in the presence of chaotropic salts is known (Vogelstein, and Gillespie, 1979). According to this method, using high concentrations of chaotropic salts, such as sodium iodide, sodium perchlorate, or guanidine thiocyanate, DNA is isolated and purified from agarose gels and RNA and DNA preparations are isolated and purified from various extracts (Boom et al. 1990; Yamado et al., 1990).
Although the physical processes resulting in an adsorption of the nucleic acids on mineral substrates in the presence of chaotropic reagents are not understood in detail, it is believed that the reason of this adsorption lies in disturbances of higher-order structures of the aqueous medium. This leads to adsorption or denaturation of the dissolved nucleic acid on the surface of the glass or silica-gel particles. In the presence of high concentrations of chaotropic salts, this adsorption will occur almost quantitatively. Elution of the adsorbed nucleic acids is performed in the presence of buffers having low ionic strengths (salt concentrations).
Prior methods allow for the isolation of nucleic acids and fragments thereof using glass, silica, zeolite, or diatomaceous earth (Little, U.S. Pat. No. 5,075,430; Boom, U.S. Pat. No. 5,234,809). These methods may be used in a form with filters or columns, or as magnetically responsive particles (hereinafter, “magnetic particles”) to expedite separations. For example, (Smith, U.S. Pat. No. 6,027,945) teaches the purification of RNA and DNA from silica magnetic beads.
Silica magnetic particles have limited utility in some samples. For example, the inventors have found that silica beads cannot efficiently isolate RNA from certainly biosamples, such as human plasma. Thus there exists a need for other support chemistries that can bind and purify RNA in manner that is adaptable to streamlined procedures, particularly for automated high throughput sample preparation.
Hawkins (U.S. Pat. No. 5,705,628) teaches that carboxy-coated magnetic particles can be used to isolate DNA, but provides no examples or data supporting the use of such particles for the purification of RNA. Moreover, these particles are chemically distinct from dextran-modified particles, which do not contain carboxy groups.
Nargessi and Pourfarzaneh (U.S. Patent Application Publication 2003/0092045) appear to disclose the use of cellulose particles or cellulose paper in the isolation and purification of nucleic acids such as DNA, RNA, and PNA. A combination of PEG and NaCl is suggested to allow binding of the nucleic acids, which can then be purportedly eluted in water or TE buffer. However, the application provides no examples or data supporting the use of such particles for the purification of RNA. Additionally, the application appears to report the purification of DNA by Sephadex G-25, although no binding buffers other than those containing PEG and NaCl are described. The paper does not appear to teach or suggest anything about the purification of nucleic acids, and RNA in particular, using magnetic particles.
Long chain carbohydrate molecules have been used to precipitate nucleic acids. For example, Dextran and glycogen have been used as co-precipitants for DNA. Polyethyleneglycol (PEG), in combination with appropriate salt concentrations, induces nucleic acid precipitation. Moreover, PEG/salt conditions also enable DNA purification using carboxy-modified (U.S. Pat. No. 5,705,628) and cellulose particles (U.S. Patent Application Publication 2003/0092045).
Dextran describes a family of polysaccharides that are produced by bacteria when grown on a sucrose substrate. Dextran is a glucose polymer linked primarily by α(1→6) bonds. A number of dextrans exist, and these are differentiated by the extent of branching and polymer chain length of the α-D-glucopyranosyl monomers. Dextrans are distinguished by their average molecular weight; for example, Dextran 40 has an average molecular weight of 40,000, whereas Dextran 75 has an average molecular weight of 75,000. Historically, Dextran has been used in the food industry, and therapeutically as a plasma volume expander and blood flow adjuvant. Dextran has also found use for emerging clinical applications of magnetic microparticles; dextran is a preferred coating for such particles owing to its biocompatibility and biodegradability, and its abundance of chemical “handles” (hydroxyl groups) that can be functionalized with a range of different chemistries. In this application, the dextran-coated particles may be used as a magnetic carrier for target drug delivery. For example, Micromod Partikeltechnologie GmbH sells several dextran-modified particles that are 50 nm to 250 nm in size for purposes of drug targeting or MRI contrast imaging. Two of these particles (Nanomag® Dextran (plain) and Nanomag® Dextran-SO3H) are described herein in the context of the invention. Advanced Magnetics, Inc., offers a Dextran-modified magnetic particle termed Combidex® that has found utility as a localizing agent for the diagnostic imaging of lymph nodes.
In contrast, applications for dextran-modified particles, specifically magnetic dextran-modified particles, in nucleic acid purification from biosamples are lacking. Although a crosslinked dextran particle known as Sephadex (Amersham Biosciences) is currently used to purify DNA in various molecular biology protocols, this purification is accomplished by size exclusion in a non-organic, non-chaotropic solution that does not require specific binding interactions between DNA and the non-magnetic Sephadex particle itself. Indeed, the application of choice for Sephadex in DNA purification is to allow the facile separation of unincorporated (“free”) deoxynucleotides from the target DNA polymer that is free from biological contaminants. No embodiments for the use of Sephadex exist for the efficient isolation of nucleic acid from biosamples.
Magnetic particles modified with PEG are commercially available. Micromod Partikeltechnologie GmbH sells several magnetic particles of various sizes that have been modified with PEG-300; that is, PEG polymers with an average molecular weight of 300 g/mol. However, there appears to be no prior use of these particles for nucleic acid purification.
In view of the above, there is a need for nucleic acid purification protocols that overcome some or all of the problems incumbent in the state of the art at the time of the filing of this application.